Capillary array electrophoresis is the method of choice for fast reliable DNA sequencing, or other DNA analysis, using automated, commercial sequencing instruments. However, the read length per capillary run that these instruments can deliver is typically no longer than 600-800 bases. To increase the read length further, the separation polymer matrix as well as the separation conditions need to be improved.
It is well known that an elevated column temperature helps to resolve compressions and to increase read length by relaxing stretched DNA in the applied field. An optimum separation of 1000 bases with 97% accuracy has been achieved at 150 V/cm and a column temperature of 50xc2x0 C. with a matrix containing 2% w/w of a high molecular mass LPA (Carrilho et al., Anal. Chem. 68:3305-3313, 1996). If elevations in column temperature for a sequencing run beyond 50xc2x0 C. were obtainable, even longer read DNA sequencing lengths should be achievable. However, the thermal stability of the polymer solution network puts limits on the maximum possible column temperature for sequencing runs.
We have determined that a blend of two hydrophilic polymers (e.g., polymers having alkyl residue groups, particularly linear polyacrylamides (LPA) or substituted polyacrylamides), one with high weight-average molecular mass (HMM), 5,000,000 or higher, and another with low weight-average molecular mass (LMM), 200,000 or higher, each present at a low concentration but wherein the concentration of the HMM LPA is above its entanglement threshold, forms an excellent, thermally stable matrix for long read length DNA sequencing. The molecular mass of the low molecular mass polymer is preferably up to about 15% of the mass of the high molecular mass polymer. Polymer separations using the matrix of the invention can be carried out at elevated temperatures, e.g., 70xc2x0 C., for improved selectivity in the long-base-fragment region.
In a preferred embodiment, the concentration of the low weight-average molecular mass LPA polymer is from 0.25% to 2%, the concentration of the high weight-average molecular mass LPA polymer is from 1.5% to 4% and the ratio of the concentration of the low-weight-average molecular mass LPA polymer to the concentration of the high-weight-average molecular mass LPA polymer is from 1:25 to 1:2.
Preferably, in the method of the invention, the device for conducting capillary electrophoresis is a coated capillary tube or integrated microchannel device. Preferred coatings are, e.g., covalent, adsorbed or polymer matrix adsorbed.